P 2-purinoceptor-mediated membrane currents in DDT 1 MF-2 smooth muscle cells

Abstract

The electrophysiological response evoked by ATP was investigated in the DDT 1 MF-2 smooth muscle cell line using the microelectrode technique and the whole-cell patch clamp technique. Application of ATP (10 −3 M) to the bathing solution caused a small initial depolarization of the cell membrane, followed by hyperpolarization and slow depolarization. During voltage clamping (−50 mV) a triphasic response was recorded on stimulation with ATP (10 −4-10 −3 M). A short-lasting inward current was followed by a transient outward current and a slowly decreasing inward current. This response was not affected by the receptor antagonists, propranolol (3 × 10 −6 M), phentolamine (3 × 10 −6 M), atropine (3 × 10 −6 M) or theophylline (10 −3 M). The ATP-induced currents were not modified by the voltage-dependent channel blocking agents, tetraethyl ammonium (3 × 10 −3 M), 3,4-diaminopyridine (10 −3 M), tetrodotoxin (3 × 10 −7 M) or diltiazem (10 −5 M). The fast inward current was not detectable at a low ATP concentration (10 −5 M). The outward current showed a reversal potential near −76 mV, which equals the potassium equilibrium potential. This current was abolished after neutralization of the potassium electrochemical gradient. The outward current was suppressed under calcium-free conditions and also in the presence of tolbutamide (10 −4 M) or glipizide (5 × 10 −6 M). Guanosine triphosphate (5 × 10 −6 M) promoted the outward current, while this current was inhibited in the presence of guanosine diphosphate (5 × 10 −6 M). Both inward currents persisted after reduction of the external sodium concentration or the internal chloride concentration and also in the absence of external magnesium or calcium. The fast inward current, however, declined after repeated stimulation of the cell with ATP under calcium-free conditions. The results indicate that, in DDT 1 MF-2 smooth muscle cells, ATP activates purinoceptor-operated ion channels, represented by a fast calcium current followed by a transient potassium current, this effect being calcium and GTP-dependent, and a non-specific inward current.